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Retroviral vectors have been most commonly used for performing gene function
studies in hematopoietic cells and exploited for gene therapy of hematological inherited
disorders. While unquestionably powerful, they suffer from several drawbacks including
size limitations and the ability to confer long-term, predictable levels of transgene
expression. The goal of this thesis was to develop an alternative gene transfer strategy
that may overcome some of these hurdles and further provide an improved platform for
performing gene function and gene regulation studies in hematopoietic cells. This strategy
is based on the Cre/lox recombination system and can be summarized in two steps: (i) a
simple retroviral vector is first used to introduce lox P target sites into the genome of
target cells, (ii) followed by Cre mediated integration of exogenous DNA into the
chromosomally placed lox P site. Initial studies demonstrated that site directed integration
was feasible and applicable to many cell types, as it was shown to occur in both nonhematopoietic
and hematopoietic cell lines, pluripotent embryonic stem (ES) cells, and in
hematopoietic progenitors derived from the ES in vitro differentiation system. Moreover,
this strategy is rapid, efficient and can be exploited to achieve predictable expression of a
transgene upon re-targeting a locus.
Gene regulatory mechanisms in hematopoietic cells were also studied using the
ES in vitro differentiation model. A series of human p" globin expression vectors that are
of interest for gene therapy of the hemoglobinopathies were assessed. The ES system was
shown to be a potent model as the development of erythroid progenitors during
differentiation correlated with the profile of globin gene expression and a large number of
mature erythroid cells for RNA/protein analysis were readily generated. The Cre/lox
system was shown to, be a more critical and informative method of evaluating constructs
compared to random integration, since analysis was performed at the same integration site
(assess transcription levels) and at more than one integration site (assess position effects).
Moreover, Cre mediated integration was achieved with human P globin constructs that
have been previously reported to be unstable in retroviral vectors and the size of which go
beyond the current limitations for retroviral vectors.